An altered profile of surface glycans is a well-known feature of tumor cells; however the molecular mechanisms by which specific glycans and associated glycosyltransferases regulate tumor cell behavior remain elusive. Work from our group has determined that the ST6Gal-I sialyltransferase endows ovarian cancer (OC) cells with the ability to avoid apoptosis induced by multiple stimuli including galectins, death receptor activators and chemotherapeutic drugs. In this proposal we show for the first time that ST6Gal-I protein is upregulated in the great majority of human ovarian tumors, including the lethal papillary serous adenocarcinoma subtype. Upregulation of ST6Gal-I correspondingly causes hypersialylation of selected receptors including the 1 integrin, Fas and TNFR1. In paradigm-shifting mechanistic studies, we find that ST6Gal-I-mediated sialylation of Fas and TNFR1 prevents receptor internalization, causing a shift in the signaling of these receptors toward survival rather than apoptosis. Additionally, ST6Gal-I activity confers resistance to apoptotic galectin-3 and the chemotherapeutic, cisplatin. The central hypothesis of this proposal is that ST6Gal-I overexpression underlies two crucial factors contributing to OC lethality: tumor cell resistance to platinum-based chemotherapy (Aim 1), and OC cell protection against immune stimuli within the peritoneal tumor microenvironment (Aim 2), which is rich in gal-3 as well as ligands for Fas and TNFR1. Preliminary studies show that ST6Gal-I promotes OC cell survival within patient peritoneal ascites fluid; this finding is significant in that OC cells metastasize va peritoneal transit. AIM 1: ST6Gal-I-mediated receptor sialylation in chemoresistance. We will define molecular mechanisms responsible for ST6Gal-I's role in cisplatin resistance, and determine whether receptor sialylation controls chemoresistance within the intact tumor microenvironment. Efficacy of cisplatin treatment will be evaluated for orthotopic tumors established in mice from OC cells with differential ST6Gal-I expression, and in vivo delivery of RNAi will be used to implement therapeutic knockdown of ST6Gal-I to restore cisplatin sensitivity. ST6Gal-I will be quantified in chemosensitive vs. chemoresistant human tumors to assess its prognostic potential. AIM 2: Contribution of ST6Gal-I to tumor cell survival within the peritoneal microenvironment Mechanisms underlying ST6Gal-I-dependent OC cell survival within ascites will be elucidated, with a focus on gal-3/ 1 integrin, FasL/Fas, and TNF?/TNFR1 interactions. ST6Gal-I-mediated protection of OC cells from cytotoxic immune cells will be examined, and human ovarian tumors screened for an association between ST6Gal-I and immune cell infiltration. The growth of orthotopic tumors +/- ST6Gal-I will be monitored in immunocompetent mice. The studies will have impact by showing that ST6Gal-I is a master regulatory molecule controlling tumor/ microenvironmental interactions that regulate the balance between elimination vs. persistence of OC cells that drive tumor recurrence. Innovation lies in the elucidation of: (1) novel glycosylation-dependent tumor mechanisms, and (2) a new molecular player in OC pathogenesis and biomarker for patient chemoresistance.